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  • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
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  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
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  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

• This invention was made with government support under HL141630, HL130253, HL138426, HD087351 and AR067294 from the National Institutes of Health (NIH). The government has certain rights to the invention.
• the application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format.
• The.txt file contains a sequence listing entitled “UTFDP3586WO_ST25.txt” created on June 24, 2021 and having a size of 17 kilobytes.
• the sequence listing contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety.
• the present disclosure relates to compositions and methods for the treatment or prevention of heart disease (e.g., cardiomyopathy) in a subject.
• the present disclosure relates to a vector comprising a cardiac-specific promoter operability linked to a therapeutic gene product for the treatment of heart disease (e.g., cardiomyopathy).
• Cardiomyopathy responsible for about half of cardiac-related deaths. It is estimated that about 1 in 250 to 1 in 10,000 adults are affected by some form of cardiomyopathy (McKenna et al. Circ Res. 121:722-730 (2017)). Despite major efforts in screening, diagnostics, and therapeutic strategies, the prevalence of cardiomyopathies and incidence of cardiomyopathy- related deaths remains high (Brieler Am Fam Physician. 96:640-646 (2017)).
• Cardiomyopathy refers to a collection of conditions of the heart that occur when its ability to pump blood is reduced. Reduction in proper functioning, such as a contractile dysfunction, of the heart muscle can lead to myocardial infarction, heart failure, blood clots, valve problems, and cardiac arrest. Cardiomyopathies can be separated into primary and secondary categories that result in varied phenotypes (McKenna et al. Circ Res. 121:722-730 (2017)). Primary cardiomyopathies can be genetic, acquired, or mixed in etiology. Genetic cardiomyopathies are inherited and include arrhythmogenic right ventricular dysplasia, hypertrophic, ion channel disorders, left ventricular compaction, and mitochondrial myopathies.
• Acquired cardiomyopathies are due primarily to non-secondary, non-genetic causes that lead to cardiac complications and include myocarditis, peripartum, tachycardia- induced cardiomyopathy, and stress-induced cardiomyopathy. Cardiomyopathies with mixed etiology are caused by a combination of non-genetic and genetic factors, and include dilated cardiomyopathy and restrictive cardiomyopathy. Secondary cardiomyopathies refer to heart disease resulting from an extracardiovascular cause. The underlying causes of secondary cardiomyopathies can be endocrine, infection, exposure to toxins, autoimmune related, nutritional, and/or neuromuscular.
• Cardiomyocytes play a central role cardiomyopathy pathologies. Cardiomyocytes, also called cardiac muscle cells, cardiac myocytes, or myocardiocytes, are cardiac cells that make up the heart muscle and are responsible for the contractile function that allows the heart to act as a pump. There are many mechanisms that reduce cardiomyocytes’ ability to function properly (Dadson et al. Clin Sci (Lond) 131:1375-1392 (2017)). In arrhythmogenic right ventricular cardiomyopathy, progressive replacement of cardiomyocytes with fibrotic tissue results in the electrical isolation of cardiomyocytes and atrophy of the ventricular myocardium, the major structure responsible for contractile function in the heart.
• Cardiomyopathies In mitochondrial cardiomyopathy, a deficiency in ATP production has a direct effect on contractile function in cardiomyocytes that have a high metabolic demand. Cardiomyopathies also emerge as a result of abnormal contractile function resulting from loss of normal Ca 2+ ion-release, uptake, and sequestration processes due to loss of activity in regulatory enzymes, such as sarco/endoplasmic reticulum calcium ATPase (SERCA) (Lennon et al. Int J Mol Med. 7:131- 41 (2001)).
• SERCA sarco/endoplasmic reticulum calcium ATPase
• the disclosure provides a method of treating heart failure in a subject in need thereof, the method comprising administering an effective amount of a recombinant adeno-associated vims (rAAV) virion, the rAAV virion comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWarf Open Reading Frame (DWORF) polypeptide operatively linked to a promoter.
• rAAV recombinant adeno-associated vims
• the subject suffers from or is at risk for cardiomyopathy.
• the cardiomyopathy is dilated cardiomyopathy (DCM).
• DCM dilated cardiomyopathy
• subject suffers from or is at risk for from myocardial infarction.
• the myocardial infarction is chronic myocardial infarction.
• the myocardial infarction is acute myocardial infarction.
• the rAAV virion is administered by intravenous or intracoronary injection.
• the rAAV transduces cardiac cells.
• the rAAV transduces cardiomyocytes.
• the rAAV transduction increases DWORF polypeptide expression in the heart of the subject.
• the rAAV transduction enhances SERCA activity.
• the rAAV virion is an rAAV virion of serotype AAV9.
• the AAV capsid comprises a capsid protein that shares at least 98% identity to SEQ ID NO: 14.
• the AAV capsid comprises a capsid protein shares at least 99% identity to SEQ ID NO: 14.
• the AAV capsid comprises a capsid protein comprising the polypeptide sequence of SEQ ID NO: 14.
• the promoter is a chicken cardiac troponin-T (cTnT) promoter.
• the chicken cTnT promoter comprises a polynucleotide sequence that shares at least 95% identity to SEQ ID NO: 11.
• the chicken cTnT promoter comprises a polynucleotide sequence that shares at least 98% identity to SEQ ID NO: 11.
• the chicken cTnT promoter comprises the polynucleotide sequence of SEQ ID NO: 11.
• DWORF polypeptide is mouse DWORF polypeptide.
• the DWORF polypeptide comprises a polypeptide sequence that shares at least 95% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
• the DWORF polypeptide comprises a polypeptide sequence that shares at least 98% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
• the DWORF polypeptide comprises the polypeptide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
• the expression cassette is flanked by AAV inverted terminal repeats (ITRs).
• ITRs are AAV2 ITRs.
• the ITRs comprise the polynucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13.
• the subject experiences improved symptoms associated with MCD following administration.
• the improved symptoms are one or more of enhanced contractility; reduced fatigue; reduced dyspnea; reduced edema; reduced chest pain; reduced arrhythmias; reduced blood clots; improved heart valve function; and reduced heart murmur.
• the disclosure provides a recombinant adeno-associated vims (rAAV) virion, the rAAV virion comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWORF polypeptide operatively linked to a promoter and a pharmaceutically acceptable carrier.
• rAAV adeno-associated vims
• the rAAV virion is an rAAV virion of serotype AAV9.
• the AAV capsid comprises a capsid protein that shares at least 98% identity to SEQ ID NO: 14.
• the AAV capsid comprises a capsid protein shares at least 99% identity to SEQ ID NO: 14.
• the AAV capsid comprises a capsid protein comprising the polypeptide sequence of SEQ ID NO: 14.
• the promoter is a cardiac troponin-T (cTnT) promoter.
• the cardiac troponin-T (cTnT) promoter comprises a polynucleotide sequence that shares at least 95% identity to SEQ ID NO: 11.
• the cardiac troponin- T (cTnT) promoter comprises a polynucleotide sequence that shares at least 98% identity to SEQ ID NO: 11.
• the cardiac troponin-T (cTnT) promoter comprises the polynucleotide sequence of SEQ ID NO: 11.
• DWORF polypeptide is DWORF polypeptide.
• the DWORF polypeptide comprises a polypeptide sequence that shares at least 95% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
• the DWORF polypeptide comprises a polypeptide sequence that shares at least 98% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
• the DWORF polypeptide comprises the polypeptide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
• the expression cassette is flanked by AAV inverted terminal repeats (ITRs).
• ITRs are AAV2 ITRs.
• the ITRs comprise the polynucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 13.
• the disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising the recombinant adeno-associated virus (rAAV) virion of any one of the preceding claims and a pharmaceutically acceptable carrier.
• the composition comprises about 5xl0 13 virions.
• kits comprising a container housing the pharmaceutical composition described herein.
• FIG. 1 shows a diagram of an illustrative embodiment, an expression cassette containing polynucleotide encoding a cTnT promoter and DWORF polypeptide flanked by AAV inverted terminal repeats.
• FIG. 2A shows a western blot analysis of tissue lysates from AAV-tdTomato or AAV- DWORF treated mice 4-weeks after AAV-delivery. tdTomato expression was assessed using an antibody for red fluorescent protein (RFP). Quad, quadriceps; GP, gastrocnemius plantaris.
• FIG. 2C shows a representative hematoxylin and eosin (H&E) staining of histological sections from mice with the indicated genotypes and treatments.
• FIG. 2D shows a Western blot analysis of heart lysates from sham or MI mice treated with AAV-tdTomato or AAV-DWORF 12-weeks after surgery.
• FIG. 2F shows Masson’s trichrome staining on serial cardiac sections from mice 12- weeks after sham or MI procedures. Mice were treated with AAV-tdTomato or AAV-DWORF as indicated. Sections were taken at 0.5pm increments.
• SERCA is a calcium pump that promotes the uptake, maintenance, and cycling of Ca 2+ ions in cardiac cells, such as cardiomyocytes.
• SERCA activity is regulated by an inhibitory peptide, phospholamban.
• DWORF DWarf Open Reading Frame
• the present disclosure provides recombinant adeno-associated virus (rAAV) virions comprising a polynucleotide encoding a DWORF polypeptide, or a functional variant thereof, and methods of use thereof.
• the rAAV virions described herein may, for example, transduce cardiac cells with a polynucleotide with a sequence encoding DWORF polypeptide operatively linked to a cardiac cell-specific promoter region into the host cell genome.
• targeted cardiac cells express the DWORF polypeptide and may have increased SERCA activity.
• pharmaceutical compositions comprising the rAAV virions described herein.
• the disclosure provides methods for treating a subject diagnosed with or at risk of cardiomyopathy using the rAAV virions and pharmaceutical compositions of the disclosure.
• the rAAV virions of the disclosure may comprise an expression cassette (FIG. 1).
• the expression cassette may comprise a polynucleotide encoding a DWORF polypeptide, or functional variant thereof, optionally operatively linked to a promoter, optionally a polyadenylation signal, and optionally a transcription termination signal.
• the expression cassette may be flanked by inverted terminal repeats (ITRs). These components provide the function of expressing the transgene after a host cell is targeted by the rAAV virion.
• ITRs inverted terminal repeats
• the promoter sequence when present, controls expression of the polynucleotide encoding the DWORF polypeptide, or functional variant thereof.
• the promoter may be cell-type specific.
• Constitutive promoters are used in expression cassettes and can be, for example, the cytomegalovirus enhancer fused to the chicken b-actin promoter (CAG), simian vims 40 (SV40) promoter, and the herpes simplex virus thymidine kinase (HSV-TK) promoter (Damdindorj et al. PLoS One. 9:el06472 (2014)).
• CAG chicken b-actin promoter
• SV40 simian vims 40
• HSV-TK herpes simplex virus thymidine kinase
• Other cell-type specific promoters may also be used.
• Cardiac cell specific promoters can be, for example, the MLC2v promoter (Phillips et al. Hypertension 39:651-5 (2002)) and the cardiac Troponin-T (cTnT) promoter (Konkalmatt et al.
• the transgene polynucleotide sequence in an expression cassette can be, for example, an open reading frame encoding a protein.
• the ITRs in an expression cassette serve as markers used for viral packaging of the expression cassette (Clark et al. Hum Gene Ther. 6:1329-41 (1995)).
• the expression cassette shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16.
• the expression cassette of the present disclosure comprises a polynucleotide sequence encoding a DWORF polypeptide.
• the expression cassette provides increased expression of a DWORF polypeptide in cardiac cell.
• the cardiac cell is a cardiomyocyte.
• expression of the DWORF polypeptide may be increased 5%, 10%, 15%, 20%, or 25% compared to expression of the DWORF polypeptide factor in an untreated subject.
• expression of the DWORF polypeptide may be increased 1-fold, 2-fold, 3-fold, 4-fold, or 5- fold compared to expression of the DWORF polypeptide in an untreated subject.
• the DWORF polypeptide may be expression at any detectable level in the cardiac cell, whereas the DWORF polypeptide may be not be expressed, or expressed at undetectable levels, in an untreated subject.
• the cardiac to which the rAAV virion is administered may express a DWORF polypeptide in higher abundance than in a cardiac cell that has only endogenous (/. ⁇ ? ., native) expression of the DWORF polypeptide.
• DWORF polypeptide is an endogenous enhancer of SERCA calcium pump activity, a desirable drug target for regulation of cardiac contractility.
• DWORF is also an unusually small protein, which makes it a good candidate for delivery to a target cell or tissue by rAAV virions.
• DWORF is an endogenous protein
• expression of DWORF in humans would not be immunogenic, allowing for long-term dosing and expression.
• the structural features of DWORF polypeptides are as follows. First, the polypeptides may have 5 to 35 consecutive residues of the DWarf Open Reading Frame (DWORF), located on chromosome 3 of a mammalian species, including mouse and human (Nelson et al. Science. 351: 271-275 (2016); U.S. Pat. No. 10,570,183).
• DWORF DWarf Open Reading Frame
• the term “a peptide having no more than X consecutive residues,” even when including the term “comprising,” cannot be understood to comprise a greater number of consecutive residues.
• the peptides will be 35 residues or less, again, comprising no more than 20 consecutive residues of DWORF.
• the overall length may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
• Ranges of peptide length of 5-34/35 residues, 6-34/35 residues, 7-50 residues, 7-25, residues, 5-20 residues, 6-20 residues, 7-20 residues, and 7-15 residues are contemplated.
• the number of consecutive DWORF residues may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Ranges of consecutive residues of 5-20 residues, 5-20 residues, 6-20 residues, 7-20 residues and 5-15 residues, 5-15, residues, 6-15 residues or 7-15 residues are contemplated.
• DWORF polypeptide is human DWORF polypeptide.
• the DWORF polypeptide comprises a polypeptide sequence that shares at least 95% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
• the DWORF polypeptide comprises a polypeptide sequence that shares at least 98% identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.
• the DWORF polypeptide comprises the polypeptide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Table 2: DWORF Sequences
• the expression cassette of the disclosure comprises a promoter.
• promoter refers to a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis, i.e., a minimal sequence sufficient to direct transcription. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues and species or cell-type specific, tissue-specific, or species specific. Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors.
• enhancer sequences that may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene.
• the promoter optionally in conjunction with an enhancer, expression of the polynucleotide encoding a DWORF polypeptide, or functional variant thereof, in a target cell.
• the expression cassette comprises a cell-type specific promoter.
• the promoter specifically promotes expression of the polynucleotide encoding the DWORF polypeptide, or functional variant thereof, in a cardiac cell.
• the promoter specifically promotes expression of the polynucleotide encoding the DWORF polypeptide, or functional variant thereof, in a cardiomyocyte.
• the promoter is a chicken cardiac troponin-T (cTnT) promoter.
• the chicken cTnT promoter comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the chicken cTnT promoter (SEQ ID NO: 11).
• the expression cassette is flanked by AAV2 inverted terminal repeats (ITRs).
• ITRs comprise the polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12 and/or SEQ ID NO: 13.
• the expression cassette comprises a polyadenylation (poly(A)) signal.
• the poly (A) signal comprises the polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17.
• an rAAV virion is used to deliver the expression cassettes described herein to cardiac cells of a subject, e.g., to treat cardiomyopathy.
• the disclosure provides an rAAV virion, the rAAV virion comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWORF polypeptide operatively linked to a promoter and a pharmaceutically acceptable carrier.
• the rAAV virions of the disclosure comprise a capsid protein.
• Capsid proteins are structural proteins that make up the assembled icosahedral packaging of the rAAV virion that contains the expression cassette. Capsid proteins are classified by the serotype. Wild type capsid serotypes in rAAV virions can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 (Naso et al. BioDrugs 31:317- 334 (2017)).
• Engineered capsid types include chimeric capsids and mosaic capsids (Choi et al.
• Capsids are selected for rAAV virions based on their ability to transduce specific tissue or cell types (Liu et al. Curr Pharm Des. 21:3248-56 (2015)).
• Any capsid protein that can facilitate rAAV virion transduction into cardiac cells for delivery of a transgene, as described herein, can be used.
• Capsid proteins used in rAAV virions for transgene delivery to cardiac cells that result in high expression include AAV4, AAV6, AAV7, AAV8, and AAV9 (Zincarelli et al. Mol. Ther. 16:P1073-1080 (2008)).
• the rAAV virion is an rAAV virion of serotype AAV9.
• the AAV capsid comprises a capsid protein that shares at least 90%, 95%, 98%, 99% or 100% identity to SEQ ID NO: 14.
• the polynucleotide encoding the AAV capsid shares at least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 15.
• the AAV capsid comprises a capsid protein comprising the polypeptide sequence of SEQ ID NO: 14.
• the rAAV is replication defective, in that the rAAV virion cannot independently further replicate and package its genome.
• the DWORF polypeptide is expressed in the targeted cardiac cell, however, due to the fact that the targeted cardiac cell lacks AAV rep and cap genes and accessory function genes, the rAAV is not able to replicate.
• rAAV virions of the present disclosure encapsulating the expression cassettes as described herein can be produced using helper- free production.
• rAAVs are replication-deficient viruses and normally require components from a live helper vims, such as adenovirus, in a host cell for packaging of infectious rAAV virions.
• rAAV helper-free production systems allow the production of infectious rAAV virions without the use of a live helper virus.
• a host packaging cell line is co-transfected with three plasmids.
• a first plasmid may contain adenovirus gene products (e.g., E2A, E4, and VA RNA genes) needed for the packaging of rAAV virions.
• a second plasmid may contain required AAV genes (e.g. , REP and CAP genes).
• a third plasmid contains the polynucleotide sequence encoding the protein of interest and a promoter flanked by ITRs.
• a host packaging cell line can be, for example, AAV-293 host cells. Suitable host cells contain additional components required for packaging infectious rAAV virions that are not supplied by the plasmids.
• the CAP genes can encode, for example, AAV capsid proteins as described herein.
• the promoter is a promoter sequence as described herein.
• the promoter sequence is a cTnT promoter sequence.
• the polypeptide of interest is a DWORF polypeptide.
• rAAV virions may be used for treating disease (Wang et al. Nat Rev Drug Discov. 18:358-378 (2019)).
• rAAV virions can deliver transgenes to cells in a subject that are, in turn, expressed in the cell.
• a transgene delivered by an rAAV virion may be incorporated into the genome of the targeted cell, allowing for potential long-term expression of the transgene product.
• rAAV virions Compared to other viral transgene delivery systems, such as adenoviruses, rAAV virions have the advantage of low immunogenicity.
• rAAV virions can be used to transduce and deliver transgenes to many cells types, including eye, blood, liver, heart, joint tissue, muscle, brain kidney or lung cells (U.S. Pat. No. 10,308,957; U.S. Pat. No. 9,803,218).
• rAAV virions can contain genomes up to about 5.2 kilobases (kb), limiting the size of the polynucleotide that can be integrated into the host cell to about 4.4 kb (Choi et al. Mol Brain. 7:1 (2014)).
• rAAV virions have been used to deliver transgenes encoding polypeptides such as microdystrophin (Chamberlain et al. Mol Ther.
• VEGF vascular endothelial growth factor
• SERCA sarco/endoplasmic reticulum Ca 2+ -ATPase
• SR Ca 2+ -ATPase is a calcium ATPase-type P-ATPase.
• SERCA resides in the sarcoplasmic reticulum (SR) within muscle cells. It is a Ca 2+ ATPase that transfers Ca 2+ from the cytosol of the cell to the lumen of the SR at the expense of ATP hydrolysis during muscle relaxation. SERCA activity is necessary for proper contractile function of the heart.
• SERCA phosphorylation and nucleotide-binding domains, which form the catalytic site, and the actuator domain, which is involved in the transmission of major conformational changes.
• the rate at which SERCA moves Ca 2+ across the SR membrane can be controlled by the regulatory protein phospholamban (PLB/PLN).
• PLB regulatory protein phospholamban
• SERCA is normally inhibited by PLB, with which it is closely associated. Increased b-adrenergic stimulation reduces the association between SERCA and PLB by the phosphorylation of PLB by PKA.
• PLB regulatory protein phospholamban
• An alternative strategy to enhancing SERCA activity by delivering a SERCA2a isoform is to enhance activity of natively expressed SERCA by displacing PLB. Contacting SERCA with the DWORF polypeptide, described in detail above, can displace PLB and enhance SERCA activity.
• the present disclosure provides a method of treating heart failure in a subject in need thereof, the method comprising administering an effective amount of a recombinant adeno-associated virus (rAAV) virion, the rAAV virion comprising an AAV capsid and an expression cassette comprising a polynucleotide encoding a DWORF polypeptide operatively linked to a promoter.
• rAAV recombinant adeno-associated virus
• treating or “treatment of a condition or subject in need thereof’ refers to (1) taking steps to obtain beneficial or desired results, including clinical results such as the reduction of symptoms; (2) preventing the disease, for example, causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease, but does not yet experience or display symptoms of the disease; (3) inhibiting the disease, for example, arresting or reducing the development of the disease or its clinical symptoms; (4) relieving the disease, for example, causing regression of the disease or its clinical symptoms; or (5) delaying the disease.
• beneficial or desired clinical results include, but are not limited to, reduction of symptoms associated with heart failure, cardiomyopathy, dilated cardiomyopathy, myocardial infarction, acute myocardial infarction, and chronic myocardial infarction.
• Subjects in need of treatment using the compositions and methods of the present disclosure include, but are not limited to, a subject suffering from or being at risk of heart failure.
• a method described herein is useful to treat, for example, cardiomyopathy.
• a method described herein is useful to treat, for example dilated cardiomyopathy.
• the subject suffers from or is at risk for cardiomyopathy.
• the cardiomyopathy is dilated cardiomyopathy (DCM).
• subject suffers from or is at risk for myocardial infarction.
• the myocardial infarction is chronic myocardial infarction.
• the myocardial infarction is acute myocardial infarction.
• the methods described herein result in the reduction of one or more symptoms of a heart disease compared to the symptoms of the heart disease before administration of the rAAV virion.
• the heart diseases of the method are, but not limited to, heart failure, cardiomyopathy, dilated cardiomyopathy, myocardial infarction, chronic myocardial infarction, and acute myocardial infarction.
• symptoms include any of the diagnostic criteria or symptoms associated with heart diseases described herein. Severity and changes of symptoms and diagnostic results are determined by a medical professional qualified to deliver assessments and analyze the results of such assessments. In some embodiments of the present disclosure, symptoms are reduced following administration of the rAAVs and compositions of the disclosure.
• Common symptoms in subjects with or at risk of developing heart disease are fatigue, dyspnea, edema, chest pain, arrhythmias, blood clots, impaired heart valve function, and heart murmur.
• the subject experiences reduced symptoms associated with the heart diseases described herein following administration of the rAAV virion and compositions of the disclosure.
• the improved symptoms are one or more of enhanced contractility; reduced fatigue; reduced dyspnea; reduced edema; reduced chest pain; reduced arrhythmias; reduced blood clots; improved heart valve function; and reduced heart murmur.
• Heart contractility can be used to assess acute and chronic forms of heart failure.
• Heart contractility may be monitored by using invasive hemodynamic monitoring, continuous ECG monitoring, central venous pressure, kidney function, pulse oximetry, arterial pressure monitoring, pulmonary artery catheter, and/or transeophageal echocardiography (Kuhn C, Werdan K. Surgical Treatment: Evidence-Based and Problem-Oriented. Kunststoff: Zuckhedt; 2001. Available from: https://www.ncbi.nlm.nih.gov/books/NBK6895/).
• Dyspnea and fatigue associated with heart disease described herein can be measured using questionnaires.
• the Modified Pulmonary Functional Status and Dyspnea Questionnaire (PFSDQ-M)IO (Huang et al. Am J Crit Care. 17:436-442 (2008)) and Minnesota Fiving with Heart Failure Questionnaire (MFHFQ)ll (Bilbao et al. Health Qual Life Outcomes. 14:23 (2016)), for example, can be used to measure subjects with a heart disease as described herein.
• the questionnaires are self-administered and allow a score to be derived that is used to assess symptom severity for dyspnea, fatigue, and other heart-health related symptoms.
• Cardiomyopathy, myocardial infarction and heart valve function may be assessed using one or more of an exercise stress test, electrocardiogram, echocardiogram, chest X-ray, cardiac CT scan, or angiogram with cardiac catheterization, cardiac MRI, B-type natriuretic peptide (BNP) levels in the blood, and/or genetic screening. Further testing is required to diagnose specific types of cardiomyopathy, myocardial infarction, or heart valve dysfunction.
• an exercise stress test electrocardiogram, echocardiogram, chest X-ray, cardiac CT scan, or angiogram with cardiac catheterization, cardiac MRI, B-type natriuretic peptide (BNP) levels in the blood, and/or genetic screening.
• BNP B-type natriuretic peptide
• DCM Dilated cardiomyopathy
• Echocardiography with a PFAX view in 2D/M-mode is used to measure several paramters, including FVIDd/s, IVSd, FVPWd, and fractional shortening. These parameters are used to assess the left ventricle cavity size, wall thickness, and radial function. Diagnostic criterion for DCM includes FVIDd/s greater than 112% (2 S.D) corrected for age and body surface area (BSA). Fractional shortening less than 25% is a criterion for the diagnosis of DCM in the presence of a dilated ventricle (Mathew et al. Echo Res Pract. 4:G1-G13 (2017)).
• the rAAV virion and compositions of the present disclosure can be administered to a subject in need thereof by systemic application, e.g., by intravenous, intra-arterial or intraperitoneal delivery of a vector in analogy to what has been shown in animal models (Katz et al, Gene Ther 19:659-669 (2012)).
• the rAAV virion and compositions of the present disclosure treat or prevent heart failure.
• the cardiomyopathy wherein the vector is administered systemically.
• the rAAV virion is administered by intravenous or intracoronary injection.
• the rAAV transduces cardiac cells. In some embodiments, the rAAV transduces cardiomyocytes.
• the rAAV transduction increases DWORF polypeptide expression in the heart of the subject.
• Increased DWORF polypeptide expression typically refers to expression at least 5%, 10%, 15%, 20% or more compared to a control subject or tissue not treated with the vector.
• detectable expression means expression at 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater than a no-vector control.
• Expression can be assess by Western blot, as described in the example that follows, or enzyme-linked immunosorbent assay (ELISA), or other methods known in the art. In some cases, expression is measured quantitatively using a standard curve. Standard curves can be generated using purified protein, e.g., purified DWORF polypeptiden, by methods described in the examples or known in the art. Alternatively, expression of the therapeutic gene product can be assessed by quantification of the corresponding mRNA.
• the increased DWORF expression in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of 3xl0 14 vg/kg or less, 2xl0 14 vg/kg or less, lxlO 14 vg/kg or less, 9xl0 13 vg/kg or less, 8xl0 13 vg/kg or less, 7xl0 13 vg/kg or less, 6xl0 13 vg/kg or less, 5xl0 13 vg/kg or less, 4xl0 13 vg/kg or less, 3xl0 13 vg/kg or less, 2xl0 13 vg/kg or less, or lxlO 13 vg/kg or less.
• vg vector genomes
• the rAAV virion of the disclosure is generally delivered to the subject as a pharmaceutical composition.
• Pharmaceutical compositions comprise a pharmaceutically acceptable solvent (e.g., water, etc.) and one or more excipients.
• the pharmaceutical compositions comprise a buffer at about neutral pH (pH 5, 6, 7, 8, or 9).
• the pharmaceutical composition comprises phosphate buffered saline (e.g., PBS at pH of about 7).
• the pharmaceutical compositions may comprise a pharmaceutically acceptable salt. The concentration of the salt may be selected to ensure that the pharmaceutical composition is isotonic to, or nearly isotonic to, the target tissue.
• compositions described herein contain vehicles (e.g., carriers, diluents and excipients) that are pharmaceutically acceptable for a formulation capable of being injected.
• vehicles e.g., carriers, diluents and excipients
• saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
• dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
• Illustrative pharmaceutical forms suitable for injectable use include, e.g., sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the pharmaceutical compositions of the disclosure comprise about 1 x 10 8 genome copies per milliliter (GC/mL), about 5 x 10 8 GC/mL, about 1 x 10 9 GC/mL, about 5 x 10 9 GC/mL, about 1 x 10 10 GC/mL, about 5 x 10 10 GC/mL, about 1 x 10 11 GC/mL, about 5 x 10 11 GC/mL, about 1 x 10 12 GC/mL, about 5 x 10 12 GC/mL, about 5 x 10 13 GC/mL, or about 1 x 10 14 GC/mL of the viral vector (e.g., rAAV virion).
• the viral vector e.g., rAAV virion
• the pharmaceutical compositions of the disclosure comprise about 1 x 10 8 genome copies per milliliter (GC/mL), about 5 x 10 8 GC/mL to about 1 x 10 9 GC/mL, about 1 x 10 9 GC/mL to about 5 x 10 9 GC/mL, about 5 x 10 9 GC/mL to about 1 x 10 10 GC/mL, about 1 x 10 10 GC/mL to about 5 x 10 10 GC/mL, about 5 x 10 10 GC/mL to about 1 x 10 11 GC/mL, about 1 x 10 11 GC/mL to about 5 x 10 11 GC/mL, about 5 x 10 11 GC/mL to about 1 x 10 12 GC/mL, about 1 x 10 12 GC/mL to about 5 x 10 12 GC/mL, about 5 x 10 12 GC/mL to about 5 x 10 13 GC/mL, or about 5 x 10 13
• the pharmaceutical compositions of the disclosure comprise about 5 x 10 8 GC/mL to about 5 x 10 9 GC/mL, about 5 x 10 9 GC/mL to about 5 x 10 10 GC/mL, about 5 x 10 10 GC/mL to about 5 x 10 11 GC/mL, about 5 x 10 11 GC/mL to about 5 x 10 12 GC/mL, or about 5 x 10 12 GC/mL to about 1 x 10 14 GC/mL of the viral vector (e.g., rAAV virion).
• the viral vector e.g., rAAV virion
• the pharmaceutical compositions of the disclosure comprise about 5 x 10 8 GC/mL to about 5 x 10 10 GC/mL, about 5 x 10 10 GC/mL to about 5 x 10 12 GC/mL, or about 5 x 10 12 GC/mL to about 1 x 10 14 GC/mL of the viral vector (e.g., rAAV virion).
• the viral vector e.g., rAAV virion
• the pharmaceutical compositions of the disclosure are administered in a total volume of about 10 pL, about 20 pL, about 30 pL, about 40 pL, about 50 pL, about 60 pL, about 70 pL, about 80 pL, about 90 pL, about 100 pL, 110 pL, about 120 pL, about 130 mL, about 140 pL, about 150 pL, about 160 mL, about 170 pL, about 180 mL, about 190 pL, or about 200 pL.
• the pharmaceutical compositions of the disclosure are administered in a total volume of about 10 pL to about 20 pL, about 20 pL to about 30 pL, about 30 pL to about 40 pL, about 40 pL to about 50 pL, about 50 pL to about 60 pL, about 60 pL to about 70 pL, about 70 pL to about 80 pL, about 80 pL to about 90 pL, about 90 pL to about 100 pL, about 100 pL to 110 pL, 110 pL to about 120 pL, about 120 pL to about 130 pL, about 130 pL to about 140 pL, about 140 pL to about 150 pL, about 150 pL to about 160 pL, about 160 pL to about 170 pL, about 170 pL to about 180 pL, about 180 pL to about 190 pL, or about 190 pL to about 200 pL.
• Genome copies per milliliter can be determined by quantitative polymerase change reaction (qPCR) using a standard curve generated with a reference sample having a known concentration of the polynucleotide genome of the virus.
• the reference sample used is often the transfer plasmid used in generation of the rAAV virion but other reference samples may be used.
• the concentration of a viral vector can be determined by measuring the titer of the vector on a cell line.
• Viral titer is typically expressed as viral particles (vp) per unit volume (e.g., vp/mL).
• the pharmaceutical compositions of the disclosure comprise about 1 x 10 8 viral particles per milliliter (vp/mL), about 5 x 10 8 vp/mL, about 1 x 10 9 vp/mL, about 5 x 10 9 vp/mL, about 1 x 10 10 vp/mL, about 5 x 10 10 vp/mL, about 1 x 10 11 vp/mL, about 5 x 10 11 vp/mL, about 1 x 10 12 vp/mL, about 5 x 10 12 vp/mL, about 5 x 10 13 vp/mL, or about 1 x 10 14 vp/mL of the viral vector (e.g., rAAV virion).
• the viral vector e.g., rAAV virion
• the pharmaceutical compositions of the disclosure comprise about 1 x 10 8 viral particles per milliliter (vp/mL) to about 5 x 10 8 vp/mL, about 5 x 10 8 vp/mL to about 1 x 10 9 vp/mL, about 1 x 10 9 vp/mL to about 5 x 10 9 vp/mL, about 5 x 10 9 vp/mL to about 1 x 10 10 vp/mL, about 1 x 10 10 vp/mL to about 5 x 10 10 vp/mL, about 5 x 10 10 vp/mL to about 1 x 10 11 vp/mL, about 1 x 10 11 vp/mL to about 5 x 10 11 vp/mL, about 5 x 10 11 vp/mL to about 1 x 10 12 vp/mL, about 1 x 10 12 vp/mL to about 5 x 10 12 vp/mL,
• the present disclosure provides a kit comprising a container housing a pharmaceutical composition as described herein.
• AAV adeno-associated virus
• AAV9-cTnT-DWORF AAV-DWORF
• AAV-tdTomato control AAV9-cTnT- tdTomato viruses
• P5 postnatal day 5
• AAV-tdTomato AAV-tdTomato
• P5 postnatal day 5
• P5 postnatal day 5
• AAV-tdTomato AAV9-cTnT- tdTomato
• FIG. 2A The efficacy of AAV-DWORF gene therapy was assessed in a mouse model of DCM caused by gene deletion of muscle-specific LIM protein (MLP, encoded by the Cspr3 gene).
• AAV- DWORF may be therapeutically superior for several reasons.
• the small size of the DWORF micropeptide 34 amino acids allows it to be more efficiently translated compared to SERCA, which is a much larger multi-pass transmembrane protein (close to 1,000 amino acids).
• DWORF has a higher apparent affinity for SERCA than the inhibitory peptide phospholamban and can counteract super-inhibition of SERCA in phospholamban transgenic mice3, therefore DWORF overexpression will likely reduce the inhibition of SERCA in heart failure driven by an increased phospholamban-to- SERCA ratio (Kranias et al. Circ Res. 110:1646-1660 (2012)). Furthermore, DWORF expression itself is reduced in human heart failure and several mouse models of genetic and acquired cardiomyopathy (Makarewich el al. Elife. 7 (2018); Nelson el al. Science.
• DWORF calcium homeostasis in disease.
• This example characterizes DWORF as a molecular inotrope capable of potently enhancing SERCA 5 activity and cardiomyocyte contractility, providing additional evidence of its potential clinical relevance as a therapeutic target for heart disease.

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